Process for removing histamine to reduce loss of pla2 and melittin in bee venom

Abstract

The present invention relates to a method for removing histamine, which is a component capable of inducing an allergic reaction, from bee venom, and to a bee venom composition prepared thereby.

Description

Histamine removal process that reduces the loss of PLA2 and melittin in bee venom

[0001] The present invention relates to a method for removing histamine, a component that can cause an allergic reaction, from bee venom and a bee venom composition produced thereby.

[0002] Bee venom, which is collected from bees, has been used since ancient times to treat or alleviate various diseases and symptoms. The known effects of bee venom include the effect of suppressing pain by inhibiting the biosynthesis of prostaglandins, the effect of improving neurological disorders through nervous system stimulation, the effect of improving blood circulation through vasoconstriction and dilation, a strong anti-inflammatory effect, the effect of promoting hormone secretion through stimulation of the pituitary gland and adrenal cortex, an antibacterial effect, and the effect of improving skin.

[0003] In its raw form, bee venom is a clear, transparent, viscous liquid with a strong, bitter, aromatic taste. It has a specific gravity of 1.13, making it heavier than water, and has an acidity ranging from pH 5.2 to 5.5. The acidic content of bee venom evaporates at room temperature, leaving behind a dry matter of approximately 30%, of which approximately 75% is protein.

[0004] The main components contained in bee venom include peptides such as melittin and apamin, protein enzymes such as phospholipase A2 (PLA2) and hyaluronidase, and amines such as histamine and dopamine. It is known that the various pharmacological effects and actions of bee venom are caused by the activity of all components contained in bee venom or a single main component.

[0005] Melittin is a hemolytic and cardiotoxic peptide that accounts for approximately 40-60% of dried bee venom. It attaches to cell membranes and exhibits pharmacological effects such as hemolysis, antibacterial, and anticancer effects, and is a key component in anti-inflammatory and anti-pain effects.

[0006] Apamin is a globular peptide neurotoxin, accounting for approximately 2-3% of dried bee venom. Recent studies have shown that apamin significantly increases axon regeneration in nerve cells by promoting the production of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), which promote nerve regeneration.

[0007] PLA2, also known as lecithinase, is a phospholipid hydrolase that accounts for approximately 10-12% of dried bee venom. PLA2 is known to promote inflammation and induce neurotoxicity by inducing PGE2 production via cyclooxygenase-2 (COX2). However, recent studies have shown that PLA2 protects the brain against neurodegenerative diseases such as Parkinson's and Alzheimer's disease. Furthermore, PLA2 exerts neuroprotective and anti-inflammatory effects by inducing the activation of regulatory T cells (Tregs).

[0008] Histamine, a substance secreted by basophils and mast cells in the blood, is known to cause allergies. It dilates capillaries, increasing blood flow and activating vascular endothelial cells to promote leukocyte aggregation. Due to the histamine contained in bee venom, itching can occur immediately or several hours after bee venom administration. Furthermore, histamine can trigger anaphylaxis (shock), a severe allergic reaction that can be fatal.

[0009] As described above, bee venom contains various substances exhibiting various activities, and therefore, technologies for controlling the components in bee venom for specific purposes or removing components that may exhibit side effects have been studied. International Publication No. WO 2014 / 196674 A1 is a patent document that discloses a method for removing PLA2 (phospholipase A2) and hyaluronidase from bee venom by ultrafiltration of bee venom mixed with distilled water to remove allergens from the venom. Korean Patent Laid-Open Publication No. 10-2016-0122094 is a patent document regarding a composition for the prevention or treatment of inflammatory diseases containing purified bee venom with reduced allergens, and discloses a composition in which bee venom is mixed with distilled water and purified by a method such as column chromatography to remove PLA2 (phospholipase A2) and histamine.

[0010] However, research on a method to remove most of histamine while minimizing the loss of PLA2 and melittin, which are currently known to be useful components among the components in bee venom, is insufficient, and in particular, there has been no attempt at a specific removal process and conditions that can further preserve useful components when purifying bee venom.

[0011] Accordingly, the inventors of the present invention have invented a new method that removes most of histamine without denaturation or change of each component in bee venom through a physical method, while dramatically improving the preservation rate of useful components such as melittin and PLA2, thereby improving the production efficiency of a bee venom composition exhibiting useful activity.

[0012] [Prior Art Literature]

[0013] [Patent Document]

[0014] (Patent Document 1) International Publication No. WO 2014 / 196674 A1

[0015] (Patent Document 2) Korean Patent Publication No. 10-2016-0122094

[0016] The present invention aims to provide a method for removing histamine from bee venom, which can remove most of histamine that can cause allergies while reducing the loss of useful components such as apamin, melittin, and PLA2 from bee venom.

[0017] In addition, the present invention aims to provide a bee venom composition in which most of histamine is removed from the bee venom raw material and apamin, melittin and PLA2 are preserved in large amounts.

[0018] To achieve the above object, one aspect of the present invention provides a method for removing histamine from bee venom, including a permeation step of immersing a permeable membrane containing bee venom raw material in an alkaline solvent.

[0019] In addition, another aspect of the present invention provides a bee venom composition manufactured through the above method.

[0020] Although the effects and activities of each of the various components contained in bee venom are known, the mechanism related to the effects of bee venom is not known in detail, so the bee venom composition as a whole containing various components can be usefully utilized.

[0021] Meanwhile, among the components of bee venom, some components, such as histamine, which can cause fatal consequences when administered, need to be sufficiently removed. The histamine removal method according to the present invention has the excellent effect of removing most of the histamine from bee venom. In addition, the method of the present invention has the effect of minimizing the loss of components that exhibit useful activities while removing most of the histamine in bee venom, and in particular, the effect of suppressing the loss of components such as melittin is shown to be remarkable.

[0022] Furthermore, the method according to the present invention has the advantage of being able to exhibit high efficiency in producing a bee venom composition at a relatively low cost and ensuring stability by reducing denaturation of components within the bee venom by using a physical method.

[0023] However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0024] Figure 1 shows the histamine peak as a result of HPLC analysis.

[0025] Figure 2 shows the peaks of apamin, PLA2, and melittin as a result of HPLC analysis.

[0026] Figure 3 is a graph comparing the removal rate of histamine by HPLC after placing bee venom raw materials in a permeable membrane tube at 25°C and 4°C and permeating them with solvents of different pHs for 60, 90, 120, and 180 minutes.

[0027] Figures 4a to 4c are graphs showing the removal rates of melittin, apamin, and PLA2 by HPLC after placing bee venom raw material in a permeable membrane tube at 25°C and permeating it with solvents of different pHs for 60, 90, 120, and 180 minutes, compared to the values ​​in the pH 7 solvent as a standard (100%).

[0028] Figure 5 is a photograph of a permeable membrane tube when permeation was performed using solvents of pH 11 and pH 12 in an experiment under the same conditions as Figure 3.

[0029] Figure 6 is a graph comparing the removal rates of histamine, melittin, apamin, and PLA2 by HPLC after placing bee venom raw material in a permeable membrane tube at 25°C and permeating it with solvents of pH 9 and pH 10 for 120, 135, 150, 165, and 180 minutes.

[0030] Figure 7 is a graph comparing the removal rates of histamine, melittin, apamin, and PLA2 by HPLC after placing bee venom raw material in a permeable membrane tube at 15°C and permeating it with a solvent of pH 9 and pH 10 for 120, 135, 150, 165, and 180 minutes.

[0031] Hereinafter, the present invention will be described in detail.

[0032]

[0033] The present invention provides a method for removing histamine in bee venom.

[0034] The above method for removing histamine from bee venom includes a permeation step of immersing a permeable membrane containing bee venom raw material in an alkaline solvent.

[0035] In the present invention, the term 'bee venom (or apitoxin)' means venom coming from the ovipositor of a bee, and in some cases may refer to bee venom before undergoing the histamine removal process according to the present invention, or to bee venom after undergoing the histamine removal process, or may refer to both.

[0036] The above bee venom and / or bee venom raw material is a substance or composition that is the subject of histamine removal according to the method of the present invention, and may be obtained directly from bees or commercially available, and may also include a purified bee venom that has undergone one or more purification or processing processes.

[0037] The above bee venom and / or bee venom raw material may be derived from bees commonly referred to as honeybees, and the bees may be large honeybees (Apis dorsta), small honeybees (Apis florea), western honeybees (Apis mellifera), and / or eastern honeybees (Apis cerana), but are not limited to any type of bee that can produce bee venom.

[0038] The above bee venom and / or bee venom raw material may include histamine, apamin, melittin, and / or PLA2 (phospholipase A2).

[0039] The above-mentioned permeable membrane is a porous membrane having small pores through which substances inside and outside the permeable membrane can move. The above-mentioned permeable membrane may be manufactured and used in the form of a tube, but is not necessarily limited to a tube shape, and any shape that can contain liquid bee venom raw material inside may be applied. When the above-mentioned permeable membrane is used in the form of a permeable membrane tube, both open ends of the permeable membrane tube may be sealed, and at this time, the sealing may be performed using a tube closure, but as long as the material inside the tube cannot pass through both ends of the tube, the sealing method may be used in any way.

[0040] The permeable membrane may have a permeable molecular weight size of 0.5 kD to 1.5 kD, 0.6 kD to 1.4 kD, 0.7 kD to 1.3 kD, 0.8 kD to 1.2 kD, 0.9 kD to 1.1 kD, or 1 kD. The permeable molecular weight size may be used in the same or similar sense as MWCO (molecular weight cut off). If the permeable molecular weight size of the permeable membrane is smaller than the above range, histamine in the bee venom may not be sufficiently removed, and if it is larger than the above range, the loss of useful components such as apamin, melittin, and / or PLA2 may increase.

[0041] The above-mentioned permeable membrane may be manufactured from components of cellulose ester and / or regenerated cellulose, but is not limited thereto.

[0042] The introduction of the above bee venom raw material into the permeable membrane may involve inserting the bee venom raw material in a liquid or solution state, and more specifically, may involve dissolving the bee venom raw material in an appropriate solvent (buffer). The solvent may be the same as the alkaline solvent in which the permeable membrane is immersed, or may have a pH condition that is slightly different.

[0043] The method for removing histamine from bee venom of the present invention may further include a step of mixing the bee venom raw material with a solvent prior to placing the bee venom raw material into a permeable membrane. The step of mixing the bee venom raw material with the solvent may be performed in conjunction with ultrasonic treatment, stirring, or the like. The ultrasonic treatment may be performed for 20 to 50 minutes, 25 to 40 minutes, 25 to 35 minutes, or 35 minutes.

[0044] The method for removing histamine from bee venom of the present invention may further include a step of filtering the bee venom raw material mixed with a solvent prior to the step of placing the bee venom raw material into a permeable membrane. The filtration may be performed using, for example, a PVDF filter, but is not limited to any filtration method as long as it removes impurities from the bee venom raw material without affecting the major components (PLA2, melittin, apamin, etc.) in the venom. The PVDF filter may be a PVDF filter having a diameter of 0.4 μm to 0.5 μm or 0.45 μm.

[0045] Immersing the permeable membrane containing the above bee venom raw material in the alkaline solvent may be done by placing the permeable membrane in a container containing the alkaline solvent, and may be done by submerging part or all of the permeable membrane, and more specifically, may be done by submerging an area corresponding to the entire volume of the bee venom raw material placed in the permeable membrane so that it is in contact with the alkaline solvent with the permeable membrane in between.

[0046] The above alkaline solvent may have a pH of pH 8 or higher, pH 8.5 or higher, pH 9 or higher, pH 8 to pH 12, pH 8.5 to pH 11, or pH 9 to pH 10. The above alkaline solvent may be prepared based on distilled water, PBS, and / or physiological saline, but is not limited thereto, and may be used regardless of its type as long as it does not significantly denature or affect the activity, structure, etc. of the components in bee venom. The above alkaline solvent may be prepared by adjusting the pH by adding sodium hydroxide and / or phosphoric acid to the distilled water, PBS, and / or physiological saline, for example. More specifically, sodium hydroxide (NaOH), sodium bicarbonate (NaHCO3), ammonia (NH3), sodium carbonate (Na2CO3), potassium carbonate (K2CO3), and / or calcium oxide (CaO) may be used to prepare the alkaline solvent according to the present invention, but is not limited thereto.

[0047] In a specific embodiment of the present invention, it was confirmed that the use of an alkaline solvent has the effect of further reducing the loss of useful components (such as apamin, melittin, and PLA2) in bee venom compared to an acidic or neutral solvent (distilled water) (Fig. 3). In particular, the use of an alkaline solvent of pH 9 and pH 10 significantly reduced the loss of the useful components. For example, when the permeation step was performed at 25°C for 180 minutes, most of the histamine in bee venom was removed when an alkaline solvent of pH 9 or pH 10 was used, while the loss of useful components such as melittin and PLA2 was very small. In addition, it was confirmed that the loss prevention effect was about 18 to 25% better than the melittin removal rate when a solvent of pH 7 and pH 8.5 was used.

[0048] The method for removing histamine from bee venom of the present invention may further include a step of stirring an alkaline solvent in which a permeable membrane is submerged.

[0049] The method for removing histamine from bee venom of the present invention may further include a solvent replacement step of removing the alkaline solvent and immersing the permeable membrane in a new alkaline solvent, wherein the new alkaline solvent to be replaced may be a solvent having the same pH as the existing alkaline solvent.

[0050] The method for removing histamine from bee venom of the present invention can more specifically set the time and / or temperature conditions of the penetration step.

[0051] The above permeation step may be performed for a time of 120 to 240 minutes, 160 to 240 minutes, 165 to 240 minutes, 180 to 240 minutes, 135 to 230 minutes, 150 to 210 minutes, 160 to 200 minutes, 165 to 200 minutes, or 165 to 180 minutes. For a sufficient histamine removal effect and an effect of suppressing the loss of useful components (such as apamin, melittin, PLA2, etc.), the above permeation step may be performed for a time of 165 to 240 minutes using an alkaline solvent having a pH of 9 to 10.

[0052] In the above-mentioned permeation step, the temperature may be 20°C to 35°C, 20°C to 30°C, 23°C to 28°C, 20°C to 25°C, 25°C to 30°C, and specifically 23°C to 28°C, or 25°C. In order to achieve a sufficient histamine removal effect and an effect of suppressing the loss of useful components (such as apamin, melittin, PLA2), the above-mentioned permeation step may be performed under a temperature condition of 23°C to 28°C or 25°C using an alkaline solvent having a pH of 9 to 10.

[0053] Based on the amount of histamine in the bee venom raw material before the above penetration step, the histamine removal rate may be 85% or more, 90% or more, or 95% or more, and specifically, 95% or more. When the histamine removal method according to the present invention is performed, most of the histamine contained in the bee venom raw material can be removed. When histamine is removed according to the above removal rate range, considering that the histamine content in the initial bee venom raw material is small, the effect of removing almost all of the histamine in the bee venom can be achieved.

[0054] According to the present invention, when the histamine removal process in bee venom is performed, the preservation rate of melittin may be increased by 15% to 30%, specifically 15% to 28% or 15% to 25%, compared to the melittin preservation rate when a neutral pH is used. According to a specific embodiment of the present invention, when the permeation step was performed for 120 minutes, when alkaline solvents of pH 9 and pH 10 were used, 17.3% and 15.1% less melittin was removed, respectively, than when the neutral pH 7 was used, resulting in a melittin preservation rate of 15% or more, and when the permeation step was performed for 180 minutes, when alkaline solvents of pH 9 and pH 10 were used, 18.9% and 24.3% less melittin was removed, respectively, than when the neutral pH 7 was used, resulting in a melittin preservation rate of approximately 18% to 25%.

[0055] According to the present invention, when the histamine removal process in bee venom is performed, the preservation rate of PLA2 may be increased by 7% to 35%, specifically 7% to 34% or 7.5% to 34%, compared to the PLA2 preservation rate when neutral pH is used. The preservation rate means how much less of the component in the bee venom is removed before / after the permeation step, and the preservation rate can be calculated using the removal rate calculated based on the amount of the component in the bee venom raw material before the permeation step, and accordingly, the preservation rate can be compared by calculating whether the removal rate of components such as melittin, PLA2, and / or apamin decreases or increases. According to a specific embodiment of the present invention, when the permeation step was performed for 120 minutes, when alkaline solvents of pH 9 and pH 10 were used, 31.8% and 33.7% less PLA2 was removed, respectively, than when neutral pH 7 was used, resulting in a PLA2 preservation rate of approximately 30% to 35%. When the permeation step was performed for 180 minutes, when alkaline solvents of pH 9 and pH 10 were used, 12.4% and 7.8% less PLA2 was removed, respectively, than when neutral pH 7 was used, resulting in a PLA2 preservation rate of approximately 7.5% to 15%.

[0056] The amount of melittin and / or PLA2 in the bee venom after the penetration step can be calculated as a percentage by dividing the amount of melittin and / or PLA2 in the bee venom before the penetration step, and the preservation rate of melittin and PLA2 according to the histamine removal method of the present invention may be increased compared to the preservation rate of melittin and PLA2 according to the removal method using a neutral solvent.

[0057] Based on the amount of PLA2 in the bee venom raw material before the permeation step, the PLA2 removal rate may be 10% or less, 8% or less, or 5% or less. In addition, based on the amount of melittin in the bee venom raw material before the permeation step, the melittin removal rate may be 25% or less, 24% or less, 23% or less, or 20% or less. When the permeation is performed according to the method of the present invention, there is an advantage in that the loss of useful components such as PLA2 and melittin can be reduced because the removal rate of these components is within the above range.

[0058]

[0059] The present invention provides a bee venom composition manufactured through the above method for removing histamine from bee venom.

[0060] The above bee venom composition may include apamin, melittin, and PLA2. The above bee venom composition is characterized by having most of histamine removed and less loss of useful components (apamin, melittin, PLA2, etc.) compared to the components of raw bee venom.

[0061] The above bee venom composition may be used for, but is not limited to, pain relief, neurological disorder improvement, blood circulation improvement, anti-inflammatory, hormone secretion promotion through pituitary and adrenal cortex stimulation, antibacterial, skin improvement, and / or alleviation, prevention, or treatment of diseases or symptoms related thereto, and may be used for any of the known uses related to the efficacy and / or activity of bee venom. In particular, the above bee venom composition may be useful for use in people who are sensitive to histamine or have severe allergic reactions, and may have a reduced possibility of anaphylaxis.

[0062]

[0063] Hereinafter, the present invention will be described in detail through examples and experimental examples.

[0064]

[0065] However, the following examples and experimental examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

[0066]

[0067] [Example 1]

[0068] Analysis method for confirming the histamine removal effect according to the method of the present invention

[0069] 1-1. HPLC analysis to identify components in bee venom

[0070] Among the components contained in bee venom, standard solutions were prepared to measure the amounts of histamine, PLA2 (phospholipase A2), melittin, and apamin, and HPLC analysis was performed under the following conditions.

[0071] To prepare a histamine standard solution for quantifying histamine, first, 166 mg of a histamine standard (SIGMA-ALDRICH, H7250) was precisely measured and placed in a 50 ml flask, 40 ml of distilled water was added, and the solution was sonicated for 30 minutes and brought to the mark with a solvent. Then, 5 ml was precisely taken and placed in a 50 ml volumetric flask, brought to the mark with a solvent again, and filtered through a 0.45 ㎛ PVDF filter to prepare a histamine standard solution.

[0072] The HPLC instrument settings for histamine analysis were set as shown in Table 1 below, and HPLC analysis was performed. The HPLC analysis data for histamine are shown in Figure 1.

[0073] Item Content Analysis Method Liquid Chromatography Detector External Spectrophotometer (Measuring wavelength 210 nm) Column C18 (4.6 mm x 150 mm, 5 μm) or equivalent Column temperature 25 ± 2 ℃ Sample temperature 8 ± 2 ℃ Flow rate 1.2 ml / min Injection amount 20 μl Mobile phase pH 6.9 Buffer: Acetonitrile (85:15, v / v) (pH 6.9 buffer: Dissolve 1.7 g of potassium monophosphate, 2.85 g of potassium hydrogen phosphate trihydrate, and 0.49 g of sodium 1-decanesulfonate in 1 liter of purified water to prepare)

[0074] Additionally, to prepare a bee venom standard solution for the quantification of PLA2, melittin, and apamin in bee venom, 50 mg of bee venom was precisely measured, placed in a 10 ml flask, dissolved in distilled water, sonicated for 30 minutes, and adjusted to the mark with solvent. Then, the bee venom standard solution was prepared by filtering it through a 0.45 ㎛ PVDF filter. Bee venom was purchased from Bee Venom Lab.

[0075] HPLC instrument settings for PLA2, melittin, and apamin analysis were set as shown in Tables 2 to 4 below, and HPLC analysis was performed. The HPLC analysis data for PLA2, melittin, and apamin are as shown in Figure 2.

[0076] Item Content Analysis Method Liquid Chromatography Detector External Spectrophotometer (measuring wavelength 220 nm) Column C18 (4.6 mm x 150 mm, 5 μm) or equivalent Column Temperature 25±2 ℃ Sample Temperature 8±2 ℃ Flow Rate 2 ㎖ / min Injection Amount 50 ㎕

[0077] Time (min)Movement phase A (%)Movement phase B (%)095535208036208036.195540955

[0078] (Mobile phase A: 0.1% trifluoroacetic acid, Mobile phase B: acetonitrile:0.1% trifluoroacetic acid = 80:20)

[0079] Ingredients Relative Retention Time (RRT) Apamin 0.40 PLA ​​20 0.75 Melittin 1.00

[0080]

[0081] 1-2. Preparation of bee venom raw material test solution and permeable membrane tube

[0082] 300 mg of bee venom was precisely measured, placed in a 10 ml flask, dissolved in a solvent, sonicated for 30 minutes, and then adjusted to the mark with distilled water. Then, 5 ml was precisely taken, placed in a 50 ml volumetric flask, and 45 ml of distilled water was added to obtain a final concentration of 5 mg / ml, and filtered through a 0.45 ㎛ PVDF filter to prepare a raw bee venom test solution.

[0083] To manufacture a permeable tube containing bee venom raw materials, first, a permeable tube (Spectra / Por 6 Dialysis Tubing, 1 kD) purchased from Repligen was cut to approximately 5 cm and one side was sealed using a tube closure. 4 ml of the bee venom raw material test solution prepared as described above was added to the other side of the tube, and the air inside the permeable tube was removed as much as possible. Then, the open side of the tube was sealed using a closure (weighted closer) and placed in a container with this side facing downward to manufacture a permeable tube containing bee venom raw materials, which was used in subsequent experiments.

[0084]

[0085] [Example 2]

[0086] Confirmation of the effect of histamine removal from bee venom using a permeable membrane and alkaline solvent.

[0087] The permeable membrane tube containing the bee venom raw material manufactured in the above Example 1-2 was placed in a solvent with different pH to remove histamine, and the components of the bee venom sample remaining in the tube were analyzed by HPLC to measure and compare the amounts of histamine, PLA2, melittin, and apamin.

[0088] According to the histamine removal method of the present invention, the alkaline solvent to be used for histamine removal was prepared with a pH condition of pH 9, and the acidic solvent used as a control was prepared with a pH of 4. The pH of the solvent was adjusted using a 1 mM solution of sodium hydroxide or phosphoric acid in distilled water.

[0089] First, before the removal process using a permeable membrane, HPLC was performed on the bee venom raw material sample to measure the amounts of histamine, PLA2, melittin, and apamin contained in the bee venom raw material, and the values ​​were compared with those after the removal process. As the alkaline solvent according to the method of the present invention, solvents whose pH was adjusted to pH 8.5, pH 9, pH 10, pH 11, and pH 12 were used, and as controls, an acidic solvent of pH 5 and a neutral solvent (distilled water) of pH 7 were prepared. In addition, permeation experiments were performed under different temperature conditions, such as a low temperature condition of 4 ℃ and a neutral solvent of 25 ℃, respectively. The bee venom raw material permeable membrane tubes of Example 1-2 were placed in each solvent, and then removed for 60, 90, 120, and 180 minutes while stirring with a stirrer. Then, the permeable membrane tubes were recovered and the components in the bee venom in the tubes were analyzed by HPLC according to the method of Example 1-1. Then, the amount of each component reduced after the permeation step by the permeable membrane was compared with the initial measured amount of histamine, PLA2, melittin, and apamin in the bee venom raw material, and this was calculated as the removal rate (%) and compared.

[0090] As a result, it was confirmed that the amount of histamine before and after the permeation step was reduced and removed when the bee venom placed in the permeable membrane was permeated in a solvent according to the method of the present invention under temperature conditions of 25 ℃ (Fig. 3). The histamine removal rate was approximately 90% or more when the permeation time was 120 minutes, and the histamine removal rate was 95% or more when permeated for 180 minutes, indicating that most of the histamine in the bee venom was removed. On the other hand, in the results of the experiment performed under low temperature conditions of 4 ℃, even though the permeation was performed for 180 minutes, the histamine removal rate was very low under all pH conditions, indicating that a sufficient histamine removal effect was not observed (Fig. 3).

[0091] In addition to the histamine removal effect, the measurement results of other components at 25℃ showed that in the case of alkaline solvents (pH 8.5, 9, 10) according to the method of the present invention, not only was the histamine removal rate over 95% in 180 minutes, but the removal rates of apamin, melittin, and PLA2, which are useful components in bee venom, were significantly lower than those in acidic or neutral solvents, indicating less loss. In particular, when solvents of pH 9 and pH 10 were used, when permeated for 180 minutes, more than 95% of histamine was removed, while the removal rate of melittin was low, indicating that the loss of useful components was minimized. At 120 minutes, melittin was preserved 17.3% and 15.1% more at pH 9 and 10, respectively, compared to the neutral pH 7, and PLA2 was preserved 31.8% and 33.7% more at pH 9 and 10, respectively, compared to the neutral pH 7. Apamin was also preserved 11.8% and 22.9% more at pH 9 and 10, respectively, compared to the neutral pH 7. In addition, at 180 minutes, melittin was preserved 18.9% and 24.3% more at pH 9 and 10, respectively, compared to the neutral pH 7, and PLA2 was preserved 12.4% and 7.8% more at pH 9 and 10, respectively, compared to the neutral pH 7. In the case of Apamin, it was also found to be preserved 7.0% and 23.0% more at pH 9 and pH 10, respectively, compared to the neutral pH 7 (Figs. 4a to 4c).

[0092] Through the above results, it was confirmed that the use of an alkaline solvent according to the method of the present invention can reduce the loss of useful components, thereby having the effect of drastically reducing the production cost of raw materials.

[0093] Meanwhile, in the case of a permeable membrane using a solvent of pH 11 and pH 12, it was confirmed that the components of the bee venom coagulated within the tube, and histamine was not properly removed, making it unsuitable for use (Fig. 5).

[0094] Through the above experimental results, it was confirmed that when removing histamine from bee venom using a permeable membrane and an alkaline solvent according to the method of the present invention, histamine can be sufficiently removed while minimizing the loss of useful components such as PLA2, melittin, and apimin. It was also confirmed that the effect of preventing the loss of useful components was particularly excellent when permeating for 180 minutes or more using an alkaline solvent of pH 9 and pH 10 at 25°C.

[0095]

[0096] [Example 3]

[0097] Confirmation of the effect of pH 9 and pH 10 solvents according to additional penetration time and temperature conditions.

[0098] Through the above Example 2, it was confirmed that among the alkaline solvents according to the present invention, when using solvents of pH 9 and pH 10, the effect of removing histamine was sufficiently shown, while the effect of minimizing the loss rate of useful components such as melittin and PLA2 was the best. Therefore, the removal rate and preservation rate changes according to more specific time and temperature conditions were measured and compared using solvents of pH 9 and pH 10.

[0099] Specifically, the temperature conditions were different at 25°C and 15°C, and in the case of time conditions, the time between 120 minutes, when the histamine removal rate did not exceed 95% in Example 2, and 180 minutes, when it exceeded 95%, was further subdivided, and the amounts of histamine, melittin, apamin, and PLA2 at 120 minutes, 135 minutes, 150 minutes, 165 minutes, and 180 minutes were measured and compared. The tube manufacturing method and HPLC analysis method were performed in the same manner as in Example 1, and the removal rate confirmation process was performed in the same manner as in Example 2.

[0100] As a result of the experiment, under conditions of 25 ℃, the histamine removal rate was generally excellent as permeation proceeded for more than 120 minutes, and in particular, when permeating for 165 and 180 minutes, the histamine removal rate was over 95%, indicating that most of the histamine in the bee venom was removed. However, when using pH 9 and pH 10 solvents, the melittin removal rate was up to 21.4%, which was confirmed to be less than 25%, and the PLA2 removal rate was up to 5.2%, which was confirmed to be less than 10%, indicating that the loss of useful components was not significant (Fig. 6).

[0101] In the results of the experiment conducted under 15 ℃ temperature conditions, there were cases where the histamine removal rate did not reach 90%, and even at 165 and 180 minutes, the histamine removal rate did not reach the target value of 95%, showing that the overall histamine removal effect was lower than under 25 ℃ conditions (Fig. 7).

[0102]

[0103] Based on the experimental results of the above examples, it can be seen that when bee venom raw material is placed in a permeable membrane and the bee venom is purified using an alkaline solvent, most of the histamine that can cause allergies can be removed from the bee venom, while minimizing the loss of useful components of the bee venom, such as melittin, apamin, and PLA2. In particular, the histamine removal effect was superior at a temperature of 25°C compared to 4°C or 15°C, and among alkaline solvents, when solvents of pH 9 and pH 10 were used, most of the histamine in the bee venom was removed while the loss of useful components, such as melittin and PLA2, was low. In addition, under the above conditions, the permeation time range that best showed the desirable removal rate of histamine, melittin, and PLA2 was 165 to 240 minutes.

[0104]

[0105] Although the present invention has been described in detail only with respect to the described embodiments, it will be apparent to those skilled in the art that various modifications and variations are possible within the technical scope of the present invention, and it is natural that such modifications and variations fall within the scope of the appended claims.

Claims

1. A method for removing histamine from bee venom, comprising a permeation step of immersing a permeable membrane containing bee venom raw material in an alkaline solvent.

2. In paragraph 1, A method for removing histamine from bee venom, wherein the above-mentioned permeable membrane has a permeable molecular weight size of 0.8 kD to 1.2 kD.

3. In paragraph 1, A method for removing histamine from bee venom, wherein the above-mentioned permeable membrane is manufactured from a component selected from the group consisting of cellulose ester and regenerated cellulose.

4. In paragraph 1, A method for removing histamine from bee venom, wherein the alkaline solvent has a pH of pH 9 to pH 10.

5. In paragraph 1, A method for removing histamine from bee venom, wherein the step of immersing the above-mentioned membrane in an alkaline solvent is performed for a period of 165 to 240 minutes.

6. In paragraph 1, A method for removing histamine from bee venom, wherein the temperature in the step of immersing the above-mentioned permeable membrane in an alkaline solvent is 20°C to 35°C.

7. In paragraph 1, A method for removing histamine from bee venom, wherein the histamine removal rate is 95% or higher based on the amount of histamine in the bee venom raw material before the above-mentioned penetration step.

8. In paragraph 1, A method for removing histamine from bee venom, wherein the preservation rate of PLA2 (phospholipase A2) is increased by 15% to 30% compared to the preservation rate when a neutral solvent is used in the above-mentioned permeation step.

9. In paragraph 1, A method for removing histamine from bee venom, wherein the preservation rate of melittin is increased by 7% to 35% compared to the preservation rate when a neutral solvent is used in the above-mentioned permeation step.

10. In paragraph 1, A method for removing histamine from bee venom, further comprising a step of stirring an alkaline solvent in which a permeable membrane is submerged.

11. In paragraph 1, A method for removing histamine from bee venom, further comprising a solvent replacement step of removing the alkaline solvent and immersing the permeable membrane in a new alkaline solvent.

12. A bee venom composition manufactured by the method according to any one of claims 1 to 11.

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